A fish-specific transposable element shapes the repertoire of p53 target genes in zebrafish

Abstract

Transposable elements, as major components of most eukaryotic organisms' genomes, define their structural organization and plasticity. They supply host genomes with functional elements, for example, binding sites of the pleiotropic master transcription factor p53 were identified in LINE1, Alu and LTR repeats in the human genome. Similarly, in this report we reveal the role of zebrafish (Danio rerio) EnSpmN6_DR non-autonomous DNA transposon in shaping the repertoire of the p53 target genes. The multiple copies of EnSpmN6_DR and their embedded p53 responsive elements drive in several instances p53-dependent transcriptional modulation of the adjacent gene, whose human orthologs were frequently previously annotated as p53 targets. These transposons define predominantly a set of target genes whose human orthologs contribute to neuronal morphogenesis, axonogenesis, synaptic transmission and the regulation of programmed cell death. Consistent with these biological functions the orthologs of the EnSpmN6_DR-colonized loci are enriched for genes expressed in the amygdala, the hippocampus and the brain cortex. Our data pinpoint a remarkable example of convergent evolution: the exaptation of lineage-specific transposons to shape p53-regulated neuronal morphogenesis-related pathways in both a hominid and a teleost fish.

Figure 1. Functional p53 responsive elements map within the zebrafish-specific EnSpm-N6_DR transposon.

(A) Predicted p53 responsive elements (REs) and their respective sequences in the first intron of Danio rerio trim8a long allele, trim8a short allele and trim8b. The indicated positions for REs are calculated from the annotated transcription start site. (B) Responsiveness to Danio rerio p53 transactivation of the first intron of trim8a engineered to contain different combinations of REs. The assessed mutant constructs are schematically represented on the left. (C) Zebrafish p53-dependent transactivation assessment in luciferase reporter assays of Danio rerio trim8a long allele, trim8a short allele, trim8b and Homo sapiens p21 and TRIM8.

Figure 2. Transposon-embedded p53REs show p53-mediated transactivation.

(A) Zebrafish p53-dependent transactivation assessment in luciferase reporter assays of 27 sequences containing EnSpm-N6_DR elements and mapping in close proximity to the indicated genes. Tested sequences are detailed in Supplementary Table S2, They are categorized by mapping location within gene loci, i.e. 5′, 3′, within the first or other more downstream exons. The names of the human orthologs are used for simplicity. Note that atp2b1, cacna1d, dach2, drd2, drd4, fibcd1, grin2a, itga8, lamb2, pgbd1, rics, sema3a, sh2d3c, ski, tnfsf10, ttll9 and wfikkn2 correspond to zebrafish atp2b1a, cacna1da, dacha, drd4-rs, dkeyp-51b7.3, grin2aa, zgc:172265, lamb2l, si:ch73-353p21.4, dkey-269g4.4, sema3aa, sh2d3ca, skia, tnfsf10l4, si:dkey-211h10.2 and LOC564992, respectively. The percentage of transactivated targets per mapping class is shown in panel (B).

Figure 3. trim8a and trim8b expression in zebrafish embryos.

(A) Expression pattern of trim8b at 18 hpf determined by whole mount ISH. Left panel: lateral view; right panel: dorsal view. Of note trim8a is not detected at this stage. (B) Comparison of the expression patterns of trim8a (top row) and trim8b (bottom row) after 30 to 72 hours of development. Embryos are in lateral (first three columns) or dorsal views (far right column). Trim8b expression at 48 hpf and 72 hpf (lateral view) is shown in de-yolked and flat mounted embryos (see Methods). Anterior to the left. AD, anterior diencephalon; C, cerebellum; DD, dorsal diencephalon; DH, dorsal hindbrain; H, hindbrain; OT, optic tectum; R, retina; SCN, ventral spinal chord neurons; T, tegmentum; TG, trigeminal ganglia; VMB ventro-lateral midbrain; VZ, brain ventricular zone.

Figure 4. Protein subnetwork of human orthologs of genes parasitized by EnSpm-N6_DR elements in zebrafish classified by their involvement in specific developmental pathways, such as neurogenesis, synaptic transmission and regulation of programmed cell death.

The network is visualized in STRING action view with lines and arrows of different colors indicating diverse types of interaction: binding (blue), activation (green), inhibition (red), post-translational modifications (violet) and co-expression (yellow). Of note the recently published interaction between TRIM8 and p53 was added explicitly as it is not yet annotated within the STRING database.